Keywords
cerebrospinal fluid - arachnoid granulation - dural wall - anlage
Introduction
Approximately, 60% of the human body is water.[1] Water is found in intracellular, interstitial, extracellular, or intravascular compartments
and as cerebral spinal fluid (CSF) in the brain. It wraps around the cerebrum and
the spinal cord spreads in it and flows around it. CSF is the major component of this
system.
The cerebral fluid system exhibits a dynamic structure, and intracranial fluids are
preserved in a very delicate balance in separate compartments. This delicate balance
is of great importance for the continuation of optimal neuronal function. Understanding
the dynamics related to the production, circulation, and absorption of CSF gives an
important insight into the pathophysiology of CSF-related diseases.[2] It is an important preliminary step to plan and implement treatment strategies for
related diseases. In recent years, arachnoid granulations, which were classically
known to play a critical role in CSF absorption, are under the spotlight regarding
the importance of cerebral absorption of lymphatic drainage.[3] In the present study, we aimed to investigate the histopathological presence of
arachnoid granulations, which are the major components of CSF absorption, in fetuses
and cases under 1 year of age.
Materials and Methods
The present study was discussed and approved at the meeting between the Ege University
Faculty of Medicine Ethics Committee for Clinical Research and T.C. Ministry of Justice
Forensic Medicine Institute.
The cases were randomly sampled from autopsy materials of fetuses over 26 weeks and
children under 1 year of age brought to the Macroscopic Laboratory of Ege University
Faculty of Medicine, Department of Pathology, and the Autopsy Hall of Morgue Department
of Forensic Medicine Council in Izmir for autopsy purposes. Autopsies were performed
within 24 hours of death.
The study included: four intrauterine fetal death cases over 26 weeks (two male and
two female), six autopsy cases under 1 year of age (three boys and three girls), and
one boy at 3 years of age for the control case ([Table 1]). Autopsy cases with a known pathology of the central nervous system were excluded
from the study. Autopsies were performed within the first 24 hours of death. While
the samples were taken for the study, an interauricular line was determined to pass
through the vertex in the frontal direction. Calvarium was exposed through an interauricular
incision line until the supraorbital region in the anterior and the inion in the posterior.
A vibrating dissection saw (Electronic Power Gipssage, Germany) was used in cases
younger than 1 year of age, and surgical scissors (Metzenbaum, Medikon, England) were
used in postmortem fetuses >24 weeks. Since the calvarium and dura are agglutinated
especially in fetuses, the vertex dura was removed together with the bone up to the
confluens sinuum in connection with the superior sagittal sinus, following the observation
of the calvarium from the anterior of the superior sagittal sinus on the midline.
Samples were placed and preserved in a 10% formalin solution. The materials, which
were kept in formalin for an average of 30 days, were placed on the cassettes in serial
sections from the frontal toward the caudal at 1-mm intervals. In nine samples, the
dura was preserved in a 4% acid solution before tissue monitoring since it could not
be separated from the bone. After 3 days of acid treatment, the materials were monitored
on a routine tissue tracking device in serial sections. After 16 hours, the samples
were blocked by embedding in paraffin. The blocks were cut at a 4-µm thickness on
a microtome and stained with hematoxylin and eosin ([Fig. 1]).
Table 1
Cases general characteristics
|
Case
|
Gender
|
Age
|
Cause of death
|
|
1
|
M
|
38-mo old child
|
Car accident
|
|
2
|
F
|
32-wk fetus
|
Ablation placenta
|
|
3
|
F
|
28-wk fetus
|
Ablation placenta
|
|
4
|
M
|
34-wk fetus
|
IUGR
|
|
5
|
M
|
5-mo old child
|
Sudden infant death syndrome
|
|
6
|
M
|
36-wk fetus
|
IUGR
|
|
7
|
M
|
8-mo old child
|
Sudden infant death syndrome
|
|
8
|
F
|
1-mo old child
|
Respiratory system disease
|
|
9
|
F
|
10-mo old child
|
Respiratory system disease
|
|
10
|
F
|
7-mo old child
|
Respiratory system disease
|
|
11
|
M
|
4-mo old child
|
Dehydration
|
Abbreviations: IUGR, intrauterine growth retardation.
Fig. 1 (A) Calvarium view after scalp detachment in a patient who was taken to the autopsy
table. (B) Removal of the calvarium with a dermatome. (C-D) Removal of the superior sagittal sinus with dura and calvarium.
In histological examination, all sections were evaluated for the presence of arachnoid
granulation under the microscope. Epithelial membrane antigen (EMA) was applied immunohistochemically
to three cases with suspicious structures, which were considered to be the outlines
of arachnoid granulation, as well as to the control. Five-micron thick sections obtained
from a paraffin block with EMA-suspicious structures were placed on electrostatically
charged slides (X-traTM, Surgipath Medical Industries, Richmond, IL, United States)
and dried at 50°C for 2 hours. Primary antibody Anti-EMA (clone EMA, Dako Cytomation,
1:100 dilution, catalog no: M3619) was dripped manually. Following incubation for
32 minutes at 37°C, the sections were manually cross-stained with hematoxylin and
eosin and bluing solution, dehydrated, cleared in xylene, and placed on the slices.
Results
Autopsy materials were taken from cadavers, six of which were boys and five girls.
The mean fetus age was 32 weeks, while the mean age in cases under 1 year of age was
5.8 months. One 3-year-old case was used as a control case for histological imaging.
The causes of death were as follows: ablatio placenta in two cases, intrauterine developmental
retardation in two cases, sudden infant death syndrome in two cases, respiratory disease
in three cases, secondary dehydration in one case, and car accident in one case.
In histological examination, we observed arachnoid granulations forming papillary
protrusions into the sinus, increased cellularity, and immunohistochemical EMA positivity
only in control case 1. In cases numbered 2, 3, 4, 5, 6, 8, and 11, and infantile
cases under 6 months, no structure was found to be stained in the light microscope,
consistent with the histology of arachnoid granulation ([Table 2]). In cases older than 6 months with numbers 7, 9, and 10, small papillary structures
were observed with an immature appearance, which we thought was an anlage of arachnoid
granulation. However, in the immune histochemical examination, these immature structures
demonstrated negative results for EMA staining ([Fig. 2]). Consistent with the literature, no male–female difference was observed among the
cases.
Table 2
Immunohistochemistry and AG presence table
|
Case number
|
Gender
|
Age
|
EMA
|
AG
|
|
1
|
M
|
38-mo old child
|
Implemented
|
(+)
|
|
2
|
F
|
32-wk fetus
|
Not implemented
|
None
|
|
3
|
F
|
28-wk fetus
|
Not implemented
|
None
|
|
4
|
M
|
34-wk fetus
|
Not implemented
|
None
|
|
5
|
M
|
5-mo old child
|
Not implemented
|
None
|
|
6
|
M
|
36-wk fetus
|
Not implemented
|
None
|
|
7
|
M
|
8-mo old child
|
Implemented
|
Outline of AG
|
|
8
|
F
|
1-mo old child
|
Not implemented
|
None
|
|
9
|
F
|
10-mo old child
|
Implemented
|
Outline of AG
|
|
10
|
F
|
7-mo old child
|
Implemented
|
Outline of AG
|
|
11
|
M
|
4-mo old child
|
Not implemented
|
None
|
Abbreviations: AG, arachnoid granulation; EMA, epithelial membrane antigen.
Fig. 2 (A) Positive control HxE, x100 staining of case 1 (arrow indicates arachnoid granulation). (B) EMA positive picture of case 1 EMAx100 (arrow indicates arachnoid granulation). (C) Arachnoid granulation anlage HEx100 staining of case 9 (arrow pointing to the outline). (D) EMA negative picture of case 9, EMAx100 staining. EMA, epithelial membrane antigen.
The presence of EMA negativity in these structures, which were considered arachnoid
granulation outlines due to their morphological similarity, was associated with incomplete
maturation. Although these outlines were morphologically similar to the structures
of arachnoid granulation, no definitive assessment of whether they function was made.
Discussion
Since the 16th century, studies have been performed by various researchers in different
science institutions around the world, from clinicians such as Cushing and Dandy to
physiologists such as Weed, to understand the physiology of CSF circulation and absorption.[4] Understanding and resolving this delicate balance is vital for establishing pathophysiology
and treatment models of CSF-related diseases.
Arachnoid granulation is classically thought of as the main component of CSF reabsorption.
Vesalius and Willis are known for their studies conducted in the 16th and 17th centuries
to establish the structure of arachnoid granulations. However, in 1721, Pacchioni
was the first to describe his views on the sectarian roles of lacuna around the superior
sagittal sinus in dissection studies. In the late 19th century, Luschka mentioned
that the arachnoid structures were invaginated with the superior sagittal sinus.[5] Quincke injected sulfide (red mercury) into the CSF in animal models and showed
that the material distributed around the superior sagittal sinus under the microscope,
mentioning that the villi herein play a role in CSF circulation. In 1875, Key and
Retzius demonstrated the role of choroid plexus in CSF drainage and investigated the
infusion of gelatin in human cadaver samples in which they showed that this substance
entered villous structures and then switched to lateral lacunae, passing through venous
sinuses and CSF circulating back into the blood. They also found stained substance
in cervical lymph nodes.[6] In 1901, Cushing mentioned one-way absorption of arachnoid projections of CSF, and
then, in 1914, Weed conducted further studies on this subject, reporting that these
structures represented a semipermeable blind diverticulum interspersed between the
venous blood in the cerebral sinuses and the CSF in the subarachnoid space.[7]
[8]
The revision of the old information about CSF flow was first started with the studies
by Welch et al. Welch and Friedman first focused on Cushing's work on arachnoid villi
and ultimately found that the statements were valid. Perfusion studies on cranial
arachnoid villus in the green monkey form the basis of these studies. Their experiments
demonstrated that a kind of balance allows the passage of cerebrospinal fluid as well
as some proteins and particles from the arachnoid villi to the cranial sinuses.[9] In the classical imaging methods, arachnoid granulation is usually seen in humans
after the first 12 months of life, while it is not observed in earlier infants and
animal models.[7]
[10] Similar to our histological study, Gomez et al evaluated 27 human fetuses and newborns
by removing the superior sagittal sinus and confluens sinuum in the postmenstrual
26 to 54 weeks period. However, unlike our study, they evaluated electron microscopy
images, not light microscopy. In this study, they observed oval depressed areas on
the sinus wall in 26-week fetuses and reported that arachnoid tissue was histologically
clustered on the dural wall. They reported that the recesses in the wall became more
irregular at week 30 and that arachnoid granulations were observed under the electron
microscope at week 39.[11]
In our study, papillary protrusions into the sinuses, increased cellularity, and arachnoid
granulation structures showing EMA positivity were monitored immunohistochemically
in only one control case. No structure was found to be stained in the light microscope
compatible with the histology of arachnoid granulation in intrauterine and infantile
cases under 6 months. Small papillary structures with an immature appearance, which
were thought to be arachnoid granulation outlines, were observed in cases older than
6 months. However, in the immune histochemical examination, these immature structures
demonstrated negative results for EMA staining. The presence of EMA negativity in
these structures, which were considered arachnoid granulation outlines due to their
morphological similarity, was associated with incomplete maturation.
In the literature, there are a limited number of studies mentioning the morphology
of human arachnoid villi and granulations. In the studies conducted by Le Gros, Ferner,
and Thomas at different times, as in our study, it was mentioned that arachnoid cell
clusters that can be observed in the early fetus will be the precursors of arachnoid
villi and granulations that will form in the later stages of life. In our study, no
male–female difference was found between the cases, which was consistent with the
literature. Available studies on this subject are generally related to the size or
location of arachnoid granulations. Our study aimed to evaluate arachnoid villi, which
are of great importance for CSF absorption and are considered to have completed their
morphological maturation. Arachnoid villi were observed to be structures completing
maturation and forming finger-like projections into the superior sagittal sinus in
our 36-month postnatal control case. Available images of arachnoid villi and granulation
during fetal and early infancy in the literature may show parallelism with the structures
that we considered to be arachnoid granulation patterns. However, we think that granulation
with the function of functional absorption within the first postnatal year is out
of the question.
In the treatment of hydrocephalus, ventriculoperitoneal (VP) shunt insertion is associated
with a high failure rate and many complications in early infancy and premature infants,
as in every period of life.[12]
[13] In the light of studies on hydrocephalus, E3V (endoscopic third ventriculostomy)
is a good alternative in the treatment of obstructive, noncommunicative hydrocephalus,
especially in cases of aqueduct stenosis. However, E3V is still a controversial treatment
option in young children, especially infants younger than 6 months. In their study
with 23 patients in 2009, Ogiwara et al mentioned that age is an important prognostic
factor for E3V, but hydrocephalus etiology bears more significance when planning surgery.[14] In 2006, DiRocco compared shunt surgery and E3V, reporting that there was no difference
between these two surgical procedures in terms of complications.[15]
In 2004, Koch and Wagner mentioned that CSF reabsorption disorders are one of the
minor effective causes of E3V failure.[16] Available studies report varying rates of success in hydrocephalus treatment in
patients under 1 year of age. Although these varying rates are mostly associated with
the etiology of hydrocephalus, CSF resorption disorder is mentioned in a limited number
of studies. Publications are mentioning that the success of E3V is low following diseases
that impair CSF absorption in adult cases, such as a former history of meningitis
or hemorrhage.[17] After the evaluation of our study, we think that this failure in E3V treatment may
be due to the incomplete maturation of arachnoid granulations.
Conclusion
In line with the results of our study, we think that the failure after E3V intervention
in the treatment of hydrocephalus in cases under the age of 1 years may be related
to the completion of arachnoid granulation development after the 18th month of life
and the immature resorption capacity in this period. We would like to emphasize that,
in prospective studies with larger series, it may be useful to evaluate the age component
as an important parameter as well as the etiology of the disease in the treatment
planning for hydrocephalus.
